Introducing Aba-seq for Enzyme Based High-Res Mapping of Mammalian Hydroxymethylomes

New England Biolabs is well known for its extensive in house research programs – churning out numerous publications every year. The role of hydroxymethylation as a possible cancer biomarker is a topic of keen interest for all Epigenetics researchers. So, NEB researchers are especially enthused about their recent publication in Cell, along with their collaborators from Emory University School of Medicine.

In nature, AbaSI is a weapon in the arms race between bacteria and bacteriophages. Wildtype bacteriophages such as T4, are resistant to most restriction enzymes due to their 5-hmC (hydroxymethylated) and 5ghmC (glucosylhydroxymethylated) DNA. However this type of bacteriophage DNA “armor” is no match for AbaSI, or other members of a family of restriction enzymes produced by bacteria, to stop T4 replication.

NEB scientists had isolated, prepared and characterized AbaSI to show ~10,000 fold higher specificity between 5ghmC and 5-mC. This feature brought the teams closer to the goal of developing a method to explore the mammalian hydroxymethome at near single base resolution. See figure 1B for the Aba-seq method overview.

For epigenetics research, the Aba-seq method has advantages over other methods developed to analyze the hydroxymethlome at high resolution sequencing. Keep in mind, only a a tiny fraction of mammalian DNA is hydroxymethylated (~0.1%). This method is not harsh on DNA samples. Only a small amount of DNA is needed 100ng. 5HmC sites with low occupancy can be reliably detected. Also, data analysis is straightforward.

The 5hmC pattern found in mouse embryonic stem cells by the researchers suggests a dependence on TET enzymes accessibility in some areas, and a demethylation intermediate or poised epigenetic state in others. The ease of this enzymatic method certainly contrasts to the complicated nature of epigenomes!